The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods. In particular, the invention relates to a novel human stem cell growth factor-like protein.
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides xe2x80x9cdirectlyxe2x80x9d in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent xe2x80x9cindirectxe2x80x9d cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.
The present invention provides an isolated polynucleotide encoding a polypeptide having stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity. These polypeptides include those which hybridize to the complement of the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under stringent hybridization conditions, those which comprise a nucleotide sequence having greater than about 85% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33, those which comprise a nucleotide sequence having greater than about 90% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33 and those polypeptides which comprise a nucleotide sequence having greater than about 92% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33. 14. The polynucleotides may be a DNA. The present invention also encompasses polynucleotides which comprise the complement of these polynucleotides.
The present invention provides for isolated polynucleotide encoding a polypeptide having stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity with the proviso that said polynucleotide sequence does not consist of the nucleotide sequence of SEQ ID NO: 47. These polypeptides include those which hybridize to the complement of the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under stringent hybridization conditions and with the proviso that said polynucleotide sequence does not consist of the nucleotide sequence of SEQ ID NO: 47, those which comprise a nucleotide sequence having greater than about 85% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33 and with the proviso that said polynucleotide sequence does consist of the nucleotide sequence of SEQ ID NO: 47, those which comprise a nucleotide sequence having greater than about 90% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33 and with the proviso that said polynucleotide sequence does not consist of the nucleotide sequence of SEQ ID NO: 47, and those polypeptides which comprise a nucleotide sequence having greater than about 92% sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33 and with the proviso that said polynucleotide sequence does not consist of the nucleotide sequence of SEQ ID NO: 47.
The present invention provides for an isolated polynucleotide that comprises the mature protein coding sequence of SEQ ID NO: 9, 11, 12,31 or 33. The invention also provides for an isolated polynucleotide that comprises the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33.
The invention provides for a DNA encoding a polypeptide having stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity wherein the encoded polypeptide has an amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34; or the encoded polypeptide has an amino acid sequence including deletion, substitution or insertion of one or several amino acids in the amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34, and which has an activity to support proliferation or survival of hermatopoietic stem cell or hematopoietic progenitor cell, with a proviso that C-terminal amino acid sequence does not comprise the amino acid sequence of SEQ ID NO: 46.
The invention provides for a DNA encoding comprising the nucleotide stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity, which is a DNA which comprises at least nucleotides 574 to 1347 of SEQ ID NO: 31; or a DNA which is hybridizable with the nucleotide sequence of SEQ ID NO: 31 or a probe prepared from said sequence, under stringent conditions, and which has an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell. These include DNAs which hybridize under the following stringent conditions: 6xc3x97SSC,5xc3x97Denhardt, 0.5% SDS and 68xc2x0 C. (SSC 3M NaCl, 0.3M sodium citrate, 50xc3x97Denhardt/1% BSA/1% polyvinyl pyrrolidone, 1% Ficoll 400/, or 6xc3x97SSC, 5xc3x97Denhardt, 0.5% SDS, 50% formamide and 42xc2x0 C.
The invention provides for a DNA encoding a polynucleotide encoding a polypeptide having stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity, which is a DNA which comprises at least nucleotides 321 to 1074 of SEQ ID NO: 33; or DNA which is hybridizable with the nucleotide sequence of SEQ ID NO: 33 or a prove prepared from said sequence, under stringent conditions, and which has an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell. These include DNAs which hybridize under the following stringent conditions: 6xc3x97SSC/5xc3x97Denhardt, 0.5% SDS and 68xc2x0 C. (SSC 3M NaCl, 0.3M sodium citrate, 50xc3x97Denhardt/1% BSA/1% polyvinyl pyrrolidone, 1% Ficoll 400, or 6xc3x97SSC, 5xc3x97Deanhardt, 0.5% SDS, 50% Formamide and 42xc2x0 C.
The invention also provides for vectors, including expression vectors, comprising the polynucleotide of the present invention. The invention futher provides for host cells genetically engineered to express a polynucleotide of the present invention. The invention provides for host cells genetically engineered to contain a polynucleotide of the present invention in operative association with a regulatory sequence that controls expression of the polynucleotide in the host cell. These host cells include those which have been genetically engineered to contain a heterologous regulatory sequence that increases expression of an endogenous polynucleotide.
The invention provides for a method of producing a polypeptide having stem cell growth factor activity comprising growing these host cells in a culture medium under conditions that permit expression of said polypeptide and isolating said polypeptide from said host cell or said culture medium The invention also encompasses a polypeptide produced by this method.
The invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity. These polypeptides include polypeptides which are encoded by an isolated polynucleotide encoding a polypeptide having stem cell growth factor activity, said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein coding portion thereof, or a fragment, analog, variant or derivative thereof that encodes a polypeptide retaining stem cell growth factor activity and which hybridizes to the complement of the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 under stringent hybridization conditions.
The invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity which comprises an amino acid sequence having greater than about 85% sequence identity with the nucleotide sequence of SEQ ID NO: 10, 13, 16, 32 or 34, an amino acid sequence having greater than about 92% sequence identity with the nucleotide sequence of SEQ ID NO: 10, 13, 16, 32 or 34, with the proviso that said polypeptide sequence does not consist of the amino acid sequence of SEQ ID NO: 48.
The invention also provides for an isolated polypeptide comprising the mature protein portion of SEQ ID NO: 10, 13, 16, 32 or 34.
The invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity, wherein the polypeptide comprises one or more motifs selected from the group of a laminin-type EGF-like domain, a membrane attack complex component/perforin domain, and neurohypophysial hormone signature.
The invention provides for polypeptides which are an expression product of a DNA of the present invention, where these polypeptide which have an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell, with the proviso that the C-terminal amino acid sequence does not comprise the amino acid sequence of SEQ ID NO: 46.
The invention provides for an isolated polynucleotide that comprises the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33, which has an amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence including deletion, substitution or insertion of one or several amino acids in the amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32
The invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity polypeptide, which has an amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34, or an amino acid sequence including deletion, substitution or insertion of one or several amino acids in the amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34.
The invention also provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity, which is modified with one or more modifying agent selected from the group consisting of polyethylene glycol (PEG), dextran, poly(N-vinyl-pyrrolidone), polypropylene glycol homopolymer, copolymer of polypropylene oxide/ethylene oxide, polyoxyethylated polyol and polyvinyl alcohol.
The invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity which comprises at least ten consecutive amino acids from SEQ ID NO: 10 or 13.
The invention also provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity, which comprises at least ten consecutive amino acids from the C-terminal seventeen amino acids of SEQ ID NO: 10 or 13.
The invention provides for a polypeptide with biological activity, said polypeptide comprising at least 272 amino acids and having at least 98% identity with SEQ ID NO: 10. The invention also provides for an isolated polypeptide with stem cell growth factor activity having at least 90% identity with SEQ ID NO: 10, 13, or 16 and lacking amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
The invention also provides for an isolated polypeptide with stem cell growth factor activity having at least 90% identity with SEQ ID NO: 10, 13, or 16 and lacking any 10 consecutive amino acids from a amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
The invention provides for an isolated polypeptide with stem cell growth factor activity having at least an amino acid sequence SVSVSTVH (SEQ ID NO: 27) or VSVSTVH (SEQ ID NO: 28), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
The invention encompasses a polynucleotide which encodes any of the polypeptides of the present invention.
The invention provides for a kit comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity.
The invention further provides for a culture medium comprising an amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity polypeptide, wherein the amount is effective to maintain survival of or promote proliferation of a stem cell or germ cell.
The composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity and a pharmaceutically acceptable carrier or diluent. These compositions can be pharmaceutical compositions including those having an effect to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell, which comprises a polypeptide which has an amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34; or a polypeptide which has an amino acid sequence including deletion, substitution or insertion of one or several amino acids in the amino acid sequence comprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising at least amino acid residues 22 to 272 of SEQ ID NO: 34, and which has an activity to support proliferation or survival of hematopoietic stem cell or hematopoietic progenitor cell.
The invention provides for an antibody that binds to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activity. The antibodies of the present invention may specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34 including those which do not bind to a polypeptide having the amino acid sequence of SEQ ID NO: 48. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, antibody fragments, chimeric antibodies, and humanized antibodies. Further, the invention encompasses kits comprising the antibodies of the present invention.
The invention provides for a method for detecting a polynucleotide of the present invention in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide is detected. The invention also provides for methods for detecting a polynucleotide of the present invention in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide; and c) detecting said product and thereby the polynucleotide in the sample. These methods include a method wherein the polynucleotide detected encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or a fragment, analog, variant or derivative thereof that retains stem cell growth factor activitya polypeptide of claim 23, and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.
The invention also provides for a method for detecting a polypeptide of the present invnetion in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide is detected.
The invention also provides for a method for identifying a compound that binds to a polypeptide of the invention, comprising: a) contacting the compound with the polypeptide under conditions and for a time sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.
The invention also provides for a method for identifying a compound that binds to the polypeptide of the present invention, comprising: a) contacting the compound with the polypeptide, in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.
The invention provides for a nucleic acid array comprising a polynucleotide of the present invention or a unique segment of a polynucleotide of the present invnetion attached to a surface. These arrays include those which full-matches to the polynucleotide or a unique segment of the polynucleotide of the present inventions, those which detect mismatches to the polynucleotide or a unique segment of the polynucleotide of the present invention.
The invention provides for a method of treatment of a subject in need of enhanced activity or expression of stem cell growth factor-like polypeptide of the present invention comprising administering to the subject: (a) a composition comprising a therapeutic amount of an agonist of said polypeptide; (b) a composition comprising a therapeutic amount of the polypeptide; or (c) a composition comprising a therapeutic amount of a polynucleotide encoding the polypeptide in form and under conditions such that the polypeptide is produced; said composition comprising a pharmaceutically acceptable carrier or diluent.
The invention also provdies for a method of treatment of a subject having need of decreased activity or expression of stem cell growth factor-like polypeptide of the present invention comprising administering to the subject: (a) a composition comprising a therapeutic amount of an antagonist of said polypeptide; (b) a composition comprising a therapeutic amount of the polynucleotide that inhibits the expression of the nucleotide sequence encoding said polypeptide; and (c) a composition comprising a therapeutic amount of a polypeptide that competes with the stem cell growth factor-like polypeptide for its ligand; said composition comprising a pharmaceutically acceptable carrier or diluent.
The invention also provides for a method of supporting proliferation or survival of a stem cell or germ cell comprising contacting said cell with an amount of a polypeptide of the present invention effective to maintain survival of or promote proliferation of said cell. These methods include those wherein said cell is a primordial germ cell, germ line stem cell, embryonic stem cell, hematopoietic stem cell, hematopoietic progenitor cell, pluripotent cell, or totipotent cell. These methods also include those wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 10, 13, or 16, or comprises an amino acid sequence 90% identical to SEQ ID NO. 10, 13, or 16. These methods further include those wherein the stem cell growth factor-like polypeptide is encoded by a polynucleotide that hybridizes to the complement of a polynucleotide encoding SEQ ID NO: 10, 13, or 16 under stringent hybridization conditions.
The invention encompasses a stromal cell genetically engineered to express a polypeptide of the invention in an amount effective to support proliferation or survival of a stem cell or germ cell. These cells include primordial germ cells germ line stem cells embryonic stem cells hematopoietic stem cells hematopoietic progenitor cells pluripotent cells or totipotent cells
The invention provides for an implant comprising a cell genetically engineered to express a polypeptide of the present invention in an amount effective to support proliferation or survival of a stem cell or germ cell. These implants of the present invention include those wherein the cell is a primordial germ cell, germ line stem cell, embryonic stem cell, hematopoietic stem cell, hematopoietic progenitor cell, pluripotent cell, or totipotent cell.
The invention provides for an isolated polynucleotide comprising the protein coding cDNA insert of the plasmid deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Zip code 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 under accession number FERM BP-7198 and the mature polypeptide expressed by this polynucleotide of in a suitable host cell.
The invention also provides for an isolated polynucleotide comprising the protein coding cDNA insert of the plasmid deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Zip code 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 under accession number FERM BP-7197 and the mature polypeptide expression product expressed by this polynucleotide in a suitable host cell.
Optionally preferred are polynucleotides and polypeptides other than the nucleotide sequence set forth as SEQ ID NO: 3284 (and the polypeptide sequence encoded therein) in U.S. application Ser. No. 09/496,914 filed Feb. 3, 2000, now abandoned, and the protein set out in Genbank Accession No. BAB28811.
The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies. Specifically, the polynucleotides of the present invention are based on polynucleotides isolated from cDNA libraries prepared from human testis cells (Hyseq clone identification numbers 2880984 and 2881695), from human fetal skin (Hyseq clone identification number 15375176), adult spleen (Hyseq clone identification number 14856094), and human endothelial cells (Hyseq clone identification numbers 13804756, 13687487, 13804756).
In one aspect, the invention involves an isolated polynucleotide with stem cell growth factor activity comprising a nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 30, the mature protein coding portion thereof, the extracellular coding portion thereof, or the active domain coding portion thereof.
In one embodiment, the invention involves an isolated polynucleotide encoding a polypeptide with biological activity, and said polynucleotide hybridizes to the complement of the polynucleotide with stem cell growth factor activity under stringent hybridization conditions.
In a further embodiment, the invention involves an isolated polynucleotide encoding a polypeptide with biological activity, said polynucleotide having at least about 92% sequence identity with the polynucleotide with stem cell growth factor activity.
In a further embodiment, the invention involves an isolated polynucleotide encoding a polypeptide with biological activity, said polypeptide having greater than about 95% sequence identity with the polynucleotide with stem cell growth factor activity.
In a still further embodiment, the polynucleotide with stem cell growth factor activity is a DNA.
In another embodiment, the invention involves an isolated polynucleotide which comprises the complement of the polynucleotide with stem cell growth factor activity.
The invention also involves a vector comprising the polynucleotide with stem cell growth factor activity. Alternatively, the invention involves an expression vector comprising the polynucleotide with stem cell growth factor activity. A host cell genetically engineered to express the polynucleotide with stem cell growth factor activity is also provided. The host cell genetically engineered to contain the polynucleotide with stem cell growth factor activity in operative association with a regulatory sequence that controls expression of the polynucleotide in the host cell.
In another aspect, the invention involves an isolated polypeptide comprising an amino acid sequence consisting of SEQ ID NO: 10, 13, 16, 32 or 34, the mature protein portion thereof, the extracellular portion thereof, or active domain thereof.
Also provided is a composition comprising the polypeptide and a carrier. In another embodiment, the invention involves an antibody directed against the polypeptide. In another aspect, the invention involves a method for detecting the polynucleotide with stem cell growth factor activity in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex; and detecting the complex, so that if a complex is detected, the polynucleotide is detected.
The invention also involves a method for detecting the polynucleotide with stem cell growth factor activity in a sample, comprising contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide under such conditions; amplifying a product comprising at least a portion of the polynucleotide; and detecting said product and thereby the polynucleotide in the sample.
In a further embodiment, the polynucleotide is an RNA molecule that encodes the polypeptide, and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.
Also provided is a method for detecting the polypeptide in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and detecting formation of the complex, so that if a complex formation is detected, the polypeptide is detected.
In another embodiment, the invention provides a method for identifying a compound that binds to the polypeptide, comprising contacting the compound with the polypeptide of under conditions and for a time sufficient to form a polypeptide/compound complex; and detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.
In a further embodiment, the invention involves a method for identifying a compound that binds to the polypeptide, comprising contacting the compound with the polypeptide in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.
In another embodiment, the invention involves a method of producing the polypeptide, comprising, culturing the host cell for a period of time sufficient to express the polypeptide in said cell; and isolating the polypeptide from the cell culture or cells.
In another aspect, the invention involves a kit comprising the polypeptide. Also provided is a nucleic acid array comprising the polynucleotide or a segment of the polynucleotide attached to a surface. In a further embodiment, the array detects full-matches to the polynucleotide or a unique segment of the polynucleotide. In another embodiment, the array detects mismatches to the polynucleotide or a unique segment of the polynucleotide.
The invention also provides for a method of treatment of a subject in need thereof enhanced activity or expression of stem cell growth factor-like polypeptide comprising administering to the subject a composition selected from the group consisting of a) therapeutic amount of an agonist of said polypeptide; b) a therapeutic amount of the polypeptide; and c) a therapeutic amount of a polynucleotide encoding the polypeptide in form and under conditions such that the polypeptide is produced, and a pharmaceutically acceptable carrier. The invention also provides for a method of treatment of a subject having need to inhibit activity or expression of stem cell growth factor-like polypeptide comprising administering to the subject a composition selected from the group consisting of a) a therapeutic amount of an antagonist of said polypeptide; b) a therapeutic amount of the polynucleotide that inhibits the expression of the nucleotide sequence encoding said polypeptide; and c) a therapeutic amount of a polypeptide that competes with the stem cell growth factor-like polypeptide for its ligand, and a pharmaceutically acceptable carrier.
In another embodiment, the invention involves a polypeptide having stem cell growth factor activity comprising at least ten consecutive amino acids from SEQ ID NO: 10, 13, 16, 32 or 34. In still another embodiment, the invention involves this polypeptide comprising at least ten consecutive amino acids from the C-terminal seventeen amino acids of SEQ ID NO: 10, 13, 16, 32 or 34.
Also provided is a polypeptide with biological activity, said polypeptide comprising at least 272 amino acids and having at least 98% identity with SEQ ID NO: 10 or 34 or said polypeptide comprising at least 273 amino acids and having at least 98% identity with SEQ ID NO: 13.
In a further embodiment, the invention involves an isolated polypeptide with stem cell growth factor activity having at least 90% identity with SEQ ID NO: 10, 13, 16, 32 or 34 and lacking amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
In yet another embodiment, the invention involves an isolated polypeptide with stem cell growth factor activity having at least 90% identity with SEQ ID NO: 10, 13, 16, 32 or 34 and lacking any 10 consecutive amino acids from amino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
In another embodiment, the invention concerns a method of maintaining or promoting proliferation of a cell selected from the group consisting of primordial germ cells, germ line stem cells, embryonic stem cells, pluripotent cell, and totipotent cells, comprising contacting the cell with an effective amount of a stem cell growth factor-like polypeptide. In a further embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or comprises an amino acid sequence 90% identical to SEQ ID NO. 10, 13, 16, 32 or 34. In still a further embodiment, the stem cell growth factor-like polypeptide is encoded by a polynucleotide that hybridizes to the complement of a polynucleotide encoding SEQ ID NO: 10, 13, 16, 32 or 34 under stringent hybridization conditions.
The invention also involves an isolated polypeptide with stem cell growth factor activity having at least an amino acid sequence SVSVSTVH (SEQ ID NO: 27) or VSVSTVH (SEQ ID NO: 28), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.
In an additional embodiment, the invention concerns the polypeptide according to this invention, wherein the polypeptide comprises one or more motifs selected from the group of a laminin-type EGF-like domain, a membrane attack complex component/perforin domain, and neurohypophysial hormone signature.
The invention also encompasses any polynucleotides encoding a polypeptide according to this invention.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in the SEQ ID NO: 9, 11, 12, 31 or 33; a polynucleotide comprising any of the full length protein coding sequences of the SEQ ID NO: 9, 11, 12, 31 or 33; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of the SEQ ID NO: 9, 11, 12, 31 or 33. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in the SEQ ID NO: 9, 11, 12, 31 or 33; (b) a nucleotide sequence encoding SEQ ID NO: 10, 13-24, 32 or 34; a polynucleotide which is an allelic variant of any polynucleotides recited above; a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising SEQ ID NO: 10, 13-24, 32 or 34.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 11, 12, 31 or 33. The sequence information can be a segment of any one of SEQ ID NO: 1-7 that uniquely identifies or represents the sequence information of SEQ ID NO: 11, 12, 31 or 33. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosome. Because there are 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosome. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segment can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences in one tissue comprise approximately 5% of the entire genome sequence. Preferably, the nucleic acid fragment or subsequence comprise the twenty-one 3xe2x80x2 coding nucleotides.
Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3xc3x9725). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.
Human stem cell growth factor-like polypeptide (SEQ ID NO: 10 or 34) is approximately a 272-amino acid protein with a predicted molecular mass of approximately 30 kDa unglycosylated. The mouse homolog is set out in SEQ ID NO:32. Protein database searches with the BLAST algorithm indicate that SEQ ID NO: 10 is homologous to Mus musculus thrombospondin type 1 domain. FIG. 1 shows the alignment of polynucleotide SEQ ID NO: 9 and EST sequences SEQ ID NO: 1-7. The sequences of the present invention (SEQ ID NO: 1-12) are expected to have stem cell growth factor activity, including hematopoietic stem cell growth factor activity, as described herein.
Stem cell growth factor-like polypeptide (SEQ ID NO: 10) also has the following motifs at the designated amino acid sequence corresponding to SEQ ID NO: 10 wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:
Laminin-type EGF-like (LE) domain proteins at
100 ADCDTCFNKNFCTKCKSGFYLHL 122xe2x80x83xe2x80x83(SEQ ID NO: 17)
Vertebrate metallothioneins proteins at
92 INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANN 137xe2x80x83xe2x80x83(SEQ ID NO: 18)
Endogenous opioids neuropeptides precursors proteins at
33 MHPNVSQGCQGGCATCSDYN 52xe2x80x83xe2x80x83(SEQ ID NO: 19)
Membrane attack complex components/perforin proteins at
145 IVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQ 181xe2x80x83xe2x80x83(SEQ ID NO: 20)
HMG-I and HMG-Y DNA-binding domain proteins (Ahook) at
213 KKGRERKRKK 222xe2x80x83xe2x80x83(SEQ ID NO: 21)
HMG1/2 proteins at
198 KCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLE 239xe2x80x83xe2x80x83(SEQ ID NO: 22)
VERTEBRATE METALLOTHIONEIN SIGNATURE at
104 TCFNKNFCTKCKSG 117xe2x80x83xe2x80x83(SEQ ID NO: 23)
NEUROHYPOPHYSIAL HORMONE SIGNATURE at
148 CEVSEWNPWSPCTKKGKTCG 167xe2x80x83xe2x80x83(SEQ ID NO: 24)
Motif 100-122, a laminin-type EGF-like domain, is a component of extracellular matrix which promotes cell growth. The membrane attack complex component/perforin domain (145-185) is postulated to mediate cell-cell interaction and thus cell growth and differentiation. Neurohypophysial hormone is itself regulated by many other factors including Interleukin-1 beta and Interleukin-6. The presence of these motifs are expected in stem cell growth factor activity.
Stem cell growth factor-like protein and/or fragments or derivatives would have similar activity to stem cell growth factors and anabolic growth factors and receptors.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising SEQ ID NO: 10, 13-24, 32 or 34; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: 1-9; 11, 12, 31 or 33 or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the protein sequences listed as SEQ ID NO: 10, 13-24, 32 or 34, and xe2x80x9csubstantial equivalentsxe2x80x9d thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,.95%, 96%, 97%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.
The invention also provides compositions comprising a polypeptide of the invention. Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also provides host cells transformed or transfected with a polynucleotide of the invention.
The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.
The methods of the invention also provides methods for the treatment of disorders as recited herein which may involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies related to disorders as recited herein. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity. Specifically, methods are provided for preventing, treating or ameliorating a medical condition, including neurological diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) stem cell growth factor-like polypeptides of the invention. The mechanics of the particular condition or pathology will dictate whether the polypeptides of the invention or binding partners (or inhibitors) of these would be beneficial to the individual in need of treatment.
The invention also provides a method of promoting wound healing comprising administering a stem cell growth factor-like polypeptide of the present invention to the site of a wound or injury. The invention provides a method of promoting cell growth and morphogenesis comprising administering a stem cell growth factor-like polypeptide of the present invention to a medium of nerve cells. According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo promotion of cellular function. A polypeptide of the invention can be administered in vivo as a stem cell growth factor alone or as an adjunct to other therapies.
The invention further provides methods for manufacturing medicaments useful in the above described methods.
The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions. The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.